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Hereditary pulmonary veno-occlusive disease (hPVOD) is a severe form of autosomal recessive pulmonary hypertension and is due to biallelic loss of function of the EIF2AK4 gene (alias GCN 2) coding for GCN2. GCN2 is a stress kinase that belongs to the integrated stress response pathway (ISR). Three rat lines carrying biallelic Gcn2 mutation were generated and found phenotypically normal and did not spontaneously develop a PVOD-related disease. We submitted these rats to amino acid deprivation to document the molecular and cellular response of the lungs and to identify phenotypic changes that could be involved in PVOD pathophysiology. Gcn2 -/- rat lungs were analyzed under basal conditions and 3 days after a single administration of PEG-asparaginase (ASNase). Lung mRNAs were analyzed by RNAseq and single-cell RNAseq (scRNA-seq), flow cytometry, tissue imaging, and Western blots. The ISR was not activated after ASNase treatment in Gcn2 -/- rat lungs, and apoptosis was increased. Several proinflammatory and innate immunity genes were overexpressed, and inflammatory cells infiltration was also observed in the perivascular area. Under basal conditions, scRNA-seq analysis of Gcn2 -/- rat lungs revealed increases in two T-cell populations, a LAG3 + T-cell population and a proliferative T-cell population. Following ASNase administration, we observed an increase in calprotectin expression involved in TLR pathway activation and neutrophil infiltration. In conclusion, under basal and asparagine and glutamine deprivation induced by asparaginase administration, Gcn2 -/- rats display molecular and cellular signatures in the lungs that may indicate a role for Gcn2 in immune homeostasis and provide further clues to the mechanisms of hPVOD development.

Pulmonary fibrosis (PF) and pulmonary hypertension (PH) are chronic diseases of the pulmonary parenchyma and circulation, respectively, which may coexist, but underlying mechanisms remain elusive. Mutations in the GCN2 (general control nonderepressible 2) gene ( EIF2AK4 [eukaryotic translation initiation factor 2 alpha kinase 4]) were recently associated with pulmonary veno-occlusive disease. The aim of this study is to explore the involvement of the GCN2/eIF2α (eukaryotic initiation factor 2α) pathway in the development of PH during PF, in both human disease and in a laboratory animal model. Lung tissue from patients with PF with or without PH was collected at the time of lung transplantation, and control tissue was obtained from tumor resection surgery. Experimental lung disease was induced in either male wild-type or EIF2AK4 -mutated Sprague-Dawley rats, randomly receiving a single intratracheal instillation of bleomycin or saline. Hemodynamic studies and organ collection were performed 3 weeks after instillation. Only significant results ( P  < 0.05) are presented. In PF lung tissue, GCN2 protein expression was decreased compared with control tissue. GCN2 expression was reduced in CD31 + endothelial cells. In line with human data, GCN2 protein expression was decreased in the lung of bleomycin rats compared with saline. EIF2AK4 -mutated rats treated with bleomycin showed increased parenchymal fibrosis (hydroxyproline concentrations) and vascular remodeling (media wall thickness) as well as increased right ventricular systolic pressure compared with wild-type animals. Our data show that GCN2 is dysregulated in both humans and in an animal model of combined PF and PH. The possibility of a causative implication of GCN2 dysregulation in PF and/or PH development should be further studied.

The lack of curative options for pulmonary arterial hypertension drives important research to understand the mechanisms underlying this devastating disease. Among the main identified pathways, the platelet-derived growth factor (PDGF) pathway was established to control vascular remodeling and anti-PDGF receptor (PDGFR) drugs were shown to reverse the disease in experimental models. Four different isoforms of PDGF are produced by various cell types in the lung. PDGFs control vascular cells migration, proliferation and survival through binding to their receptors PDGFRα and β. They elicit multiple intracellular signaling pathways which have been particularly studied in pulmonary smooth muscle cells. Activation of the PDGF pathway has been demonstrated both in patients and in pulmonary hypertension (PH) experimental models. Tyrosine kinase inhibitors (TKI) are numerous but without real specificity and Imatinib, one of the most specific, resulted in beneficial effects. However, adverse events and treatment discontinuation discouraged to pursue this therapy. Novel therapeutic strategies are currently under experimental evaluation. For TKI, they include intratracheal drug administration, low dosage or nanoparticles delivery. Specific anti-PDGF and anti-PDGFR molecules can also be designed such as new TKI, soluble receptors, aptamers or oligonucleotides.

Cardiac hypertrophy, initiated by a variety of physiological or pathological stimuli (hemodynamic or hormonal stimulation or infarction), is a critical early adaptive compensatory response of the heart. The structural basis of the progression from compensated hypertrophy to pathological hypertrophy and heart failure is still largely unknown. In most cases, early activation of an inflammatory program reflects a reparative or protective response to other primary injurious processes. Later on, regardless of the underlying etiology, heart failure is always associated with both local and systemic activation of inflammatory signaling cascades. Cardiac macrophages are nodal regulators of inflammation. Resident macrophages mostly attenuate cardiac injury by secreting cytoprotective factors (cytokines, chemokines, and growth factors), scavenging damaged cells or mitochondrial debris, and regulating cardiac conduction, angiogenesis, lymphangiogenesis, and fibrosis. In contrast, excessive recruitment of monocyte-derived inflammatory macrophages largely contributes to the transition to heart failure. The current review examines the ambivalent role of inflammation (mainly TNFα-related) and cardiac macrophages (Mφ) in pathophysiologies from non-infarction origin, focusing on the protective signaling processes. Our objective is to illustrate how harnessing this knowledge could pave the way for innovative therapeutics in patients with heart failure.

Background Platelet-derived growth factor is a major regulator of the vascular remodeling associated with pulmonary arterial hypertension. We previously showed that protein widely 1 (PW1 + ) vascular progenitor cells participate in early vessel neomuscularization during experimental pulmonary hypertension (PH) and we addressed the role of the platelet-derived growth factor receptor type α (PDGFRα) pathway in progenitor cell-dependent vascular remodeling and in PH development. Methods and Results Remodeled pulmonary arteries from patients with idiopathic pulmonary arterial hypertension showed an increased number of perivascular and vascular PW1 + cells expressing PDGFRα. PW1 nLacZ reporter mice were used to follow the fate of pulmonary PW1 + progenitor cells in a model of chronic hypoxia-induced PH development. Under chronic hypoxia, PDGFRα inhibition prevented the increase in PW1 + progenitor cell proliferation and differentiation into vascular smooth muscle cells and reduced pulmonary vessel neomuscularization, but did not prevent an increased right ventricular systolic pressure or the development of right ventricular hypertrophy. Conversely, constitutive PDGFRα activation led to neomuscularization via PW1 + progenitor cell differentiation into new smooth muscle cells and to PH development in male mice without fibrosis. In vitro, PW1 + progenitor cell proliferation, but not differentiation, was dependent on PDGFRα activity. Conclusions These results demonstrate a major role of PDGFRα signaling in progenitor cell-dependent lung vessel neomuscularization and vascular remodeling contributing to PH development, including in idiopathic pulmonary arterial hypertension patients. Our findings suggest that PDGFRα blockers may offer a therapeutic add-on strategy to combine with current pulmonary arterial hypertension treatments to reduce vascular remodeling. Furthermore, our study highlights constitutive PDGFRα activation as a novel experimental PH model.

Sympathetic nervous system overdrive with chronic release of catecholamines is the most important neurohormonal mechanism activated to maintain cardiac output in response to heart stress. Beta-adrenergic signaling behaves first as a compensatory pathway improving cardiac contractility and maladaptive remodeling but becomes dysfunctional leading to pathological hypertrophy and heart failure (HF). Cardiac remodeling is a complex inflammatory syndrome where macrophages play a determinant role. This study aimed at characterizing the temporal transcriptomic evolution of cardiac macrophages in mice subjected to beta-adrenergic-stimulation using RNA sequencing. Owing to a comprehensive bibliographic analysis and complementary lipidomic experiments, this study deciphers typical gene profiles in early compensated hypertrophy (ECH) versus late dilated remodeling related to HF. We uncover cardiac hypertrophy- and proliferation-related transcription programs typical of ECH or HF macrophages and identify lipid metabolism-associated and Na + or K + channel-related genes as markers of ECH and HF macrophages, respectively. In addition, our results substantiate the key time-dependent role of inflammatory, metabolic, and functional gene regulation in macrophages during beta-adrenergic dependent remodeling. This study provides important and novel knowledge to better understand the prevalent key role of resident macrophages in response to chronically activated beta-adrenergic signaling, an effective diagnostic and therapeutic target in failing hearts.